Detection of biomarkers (e.g., nucleic acid molecules, amino acid molecules or cells) by using probes (e.g., complementary nucleic acid molecules or antibodies) having high binding affinity with the biomarkers has been routinely used in biomedical research labs and clinical diagnostics. They usually require rather long incubation time for the binding and lengthy protocols to ensure sufficient sensitivity and specificity for the detection.
The average single-base-pair hybridization and dehybridization rates in a duplex DNA formation are on the order of a millisecond. One rate limiting step, however, is for two complementary oligonucleotides to collide with each other and initiate the base pairing process. Surface-bound oligonucleotides also behave differently than oligonucleotides in a solution. A large body of literature has shown that hybridization that occurs in the interface between a solid and a solution (i.e., surface-based hybridization) exhibits substantially different hybridization kinetics than that observed in bulk solutions. Surface-based hybridization may occur via either of two mechanisms: direct hybridization from the bulk phase (i.e., 3D diffusion) or hybridization after an initial nonspecific adsorption step followed by subsequent surface diffusion to the probe (i.e., 2D diffusion). Modeling supported by experimental data has shown that the two-step mechanism involving 2D diffusion is several magnitudes faster than the direct hybridization mechanism with a proper target density and a probe concentration. In the two-step mechanism, the surface diffusion step, following the second order Langmuir model, is a rate-limiting step for efficient hybridization, since the initial adsorption are normally completed in milliseconds.
Paramagnetic agents have been used to label biomolecules for concentrating the labeled biomolecules when a magnetic field is applied. However, the paramagnetic agents are much larger than DNA probes, limiting the accessibility of labeled DNA probe to immobilized target DNA molecules and saturating the surface for hybridization. International Patent Application No. PCT/US00/14969 (published as WO 00/73506) discloses that the use of a probe attached to superparamagnetic particles having a diameter of about 1-10 nanometers reduces the hybridization time from days to minutes.
There remains a need for reliable and sensitive methods to detect biomarkers instantaneously.